Anuran males and females adopt different reproductive and behavioral strategies in different contexts. We investigated the reproductive ecology and territorial behavior of the treefrog Boana goiana (B. Lutz, 1968) from the Brazilian Cerrado. We hypothesized that competitor density/proximity would increase the behavioral responses of B. goiana males, and that mating would be assortative. We also tested if the number of eggs correlates with female size and if there is a trade-off between clutch size and egg size. We conducted two territoriality experiments to test the effects of male size, competitor proximity and competitor density. Larger males called more in the presence of a second male. In the second experiment, the largest males emitted more calls and the distance to the nearest male increased as resident males called more. In both experiments, the number of calls was influenced by either male size or spacing between males. Some males behaved as satellites, probably to avoid fights. Our analyses indicate that females choose males with similar sizes to their own, corroborating our hypothesis of size-assortative mating. We found no relationships between female size and clutch size/volume, and between egg size and number of eggs per clutch. We also report multiple spawning for this species. The low incidence of physical combats and the spacing pattern indicate that this species relies almost solely on calls to resolve contests, which could be explained by low motivation, or simply because males avoid combats to decrease injury risks. Thus, acoustic or even multimodal communication seems crucial for social interactions of B. goiana.

Clutch size, acoustic communication, assortative mating, male competition, aggressive interactions, arrival dynamics

In the classic Darwinian definition of sexual selection, in animals that reproduce sexually, males and females will adopt different strategies to ensure their reproductive success (Andersson 1994, Candolin 2019). Because their reproduction is limited in time and space (Haddad and Prado 2005, Wells 2007), anuran amphibians are good model organisms to investigate factors related to mating and reproductive success, with males and females adopting markedly different strategies (Cayuela et al. 2017, Valencia-Aguilar et al. 2020). In general terms, the reproductive success of males of many animal species can be defined by a variety of factors, including body size, territory quality, displays, and built structures (e.g. fish: McKaye et al. 1990, frogs: Olsson and Shine 1996, crabs: Murai and Backwell 2006). Specifically for male anurans, arrival at the reproductive area is often associated with mating success, and males that arrive early may increase their chances of mating (Lodé et al. 2005, Dias et al. 2017). The establishment and maintenance of a territory can also be essential for the reproductive success of anuran males (Wells 1977a). Regarding the ability to establish and maintain territories by males, acoustic signaling is an essential component of anuran social behavior (Wells 2007). Male anurans use calls to establish territories, interact aggressively with other males, maintain the spacing among calling neighbors and – most importantly – to attract females (Bastos et al. 2011, Lemes et al. 2012). The calling activity of neighbors may influence the resident males’ behavior, changing their acoustic behavior and sometimes leading to physical combats (Wells 1977a, Bastos et al. 2011, Lemes et al. 2012). Males can also assess the size of rival males by evaluating call parameters, thus avoiding a conflict with a larger opponent (Bee and Perrill 1996, Bee et al. 1999, Nali and Prado 2014). Furthermore, the establishment of a territory by one of the males can affect the outcome of the contest by increasing motivation to defend its territory (Owen and Gordon 2005).

Meanwhile, the reproductive success of females is usually affected by mate choice and their capacity to produce oocytes, which generally correlates with body size (e.g. lizards and snakes: Shine 1992, frogs: Pettitt et al. 2013). In anurans, the reproductive effort of females usually fits one of three patterns: clutches with a few large eggs, clutches with many small eggs, or variable egg sizes within the same clutch (Crump 1984, Dziminski et al. 2009, Pupin et al. 2018). Additionally, large females can produce more eggs than small females, and this positive relationship between clutch size and female size is known for several anuran species (e.g. Kusano and Hayashi 2002, Wogel et al. 2002, Dziminski and Alford 2005). In this context, the reproductive strategies of females may vary depending on the environmental conditions (Crump 1984, Pupin et al. 2018). Another important aspect is mate choice and anuran females often choose their mates (Morris 1989), favoring males that may confer them the greatest reproductive success, but strategies may vary among species (Pfennig 2000, Pettitt et al. 2013, Valencia-Aguilar et al. 2020). Within the broader framework of sexual selection, some species may exhibit assortative mating. In such cases, females may choose males with traits similar to their own, such as those with aposematic color patterns (Reynolds and Fitzpatrick 2007) or males of similar body sizes, which allows for a closer juxtaposition of the cloacae during mating, increasing fertilization rates (Licht 1976, Bastos and Haddad 1996).

Although anuran territorial behavior is well studied (Fellers 1979, Bee 2016), the vast majority of the knowledge regarding this issue comes from direct natural history observations (e.g. Oliveira et al. 2011) or experiments using playbacks (e.g. Amézquita et al. 2005). Regardless of being well established in herpetology, these approaches have some limitations: naturalistic observations depend on chance to describe some behaviors and are purely descriptive, while playback experiments are unable to address multimodal communication. In contrast, in situ experiments investigating interactions between two or more individuals are rare (e.g. Hagen et al. 2016), however, they allow for a factorial design and can be more informative than the aforementioned approaches. This becomes more important when we consider that multimodal signaling seems to be more common and important for anuran social behavior than previously thought (Hartmann et al. 2004, Richardson et al. 2010, Starnberger et al. 2014).

Boana goiana (B. Lutz, 1968) is an interesting model organism to study aspects of the reproductive biology of both male and female anurans. The species is a gladiator frog distributed in the central Brazilian states of Goiás, Minas Gerais and in the Distrito Federal (Frost 2020). In the rainy season, males of this species call perched on the vegetation surrounding water bodies (Menin et al. 2004). Furthermore, males of B. goiana are known to display territorial behavior and to be faithful to calling sites (Menin et al. 2004, Dias et al. 2017). Although some studies on the species’ biology have already been published (Bastos et al. 2003, Menin et al. 2004, Morais et al. 2016, Dias et al. 2017), little is known about the reproductive ecology and dynamics of male territoriality. Because of their territorial behavior and the presence of weaponry in males, B. goiana is particularly suitable to investigate the effect of competitor density on the reproductive and territorial behavior of hylids. Competitor density is known to affect territorial and reproductive behavior in many animal groups, such as teleost fishes (e.g. Kaspersson et al. 2010, Magellan et al. 2011), spiders (e.g. Ritschoff 2010) and passerine birds (e.g. Foltz et al. 2015). Investigations regarding the effect of competitor density in the social behavior of amphibians, conversely, focus mostly on issues such as mating success and amplexus duration (e.g. Chuang et al. 2013), with territoriality being a neglected aspect. The concept of economic defendability predicts that male competition will increase with density if the resource value outweighs the cost of defense (Grant 1993). This prediction can be applied to B. goiana: mating is restricted to the reproductive season, meaning that female availability is restricted in time, and males occupy and defend spatially limited calling perches (Dias et al. 2017). In this context, competitor density and prior establishment of territories, i.e. the resident effect (Baugh and Forester 1994), likely affect the social behavior of B. goiana. Lastly, studies that provide basic information of a species’ life history, ecology and behavior are important because not only they provide basic information for conservation purposes, but they also provide background knowledge for testing more elaborated hypotheses, and B. goiana is a potentially interesting organism to provide such information.

In the present study, we investigated the reproductive ecology and the dynamics of the territorial behavior of B. goiana, combining in-situ experiments with natural history observations. We hypothesized that: 1) because size is related to success in contests, large males will be more aggressive and will dominate interactions without a prior resident, and these contests will escalate with time; 2) because resources are restricted across time and space, an increase in competitor density will increase the intensity of aggressive behaviors displayed by resident males towards intruders; and 3) because similar body sizes facilitate cloacal juxtaposition, females will display size-assortative mate choice. We also tried to answer the following questions: a) Do large males arrive at the reproductive area early in the reproductive season? b) Is there a positive relationship between female size and clutch size? c) Is there a trade-off between egg size and number of eggs per clutch?

The study was conducted at the Floresta Nacional de Silvânia (FNS; 16°39’32”S, 48°36’29” W, elevation ca. 900 m), municipality of Silvânia, state of Goiás, central-western Brazil. The FNS is located in the Cerrado domain and includes many vegetation types, one of which is the gallery forest, where we conducted this study (sensu Oliveira-Filho and Ratter 2002). According to Koppen’s classification (Peel et al. 2007), the study area is characterized by a tropical savanna climate (Aw), with two well-defined seasons, a rainy season from October to March and a dry season from April to September.

Male anurans emit advertisement calls to attract females and announce their position to nearby males competing for the same territories and/or females (Wells 1977a). In many species, males can recognize the size and, consequently, the condition of their opponents evaluating the characteristics of the advertisement calls, which allows males to avoid fights they have low probabilities to win (Wells 1978, Robertson 1986, Bee and Perril 1996, Nali and Prado 2014). Thus, when body condition asymmetries between males are large and more easily assessed, less intense encounters are expected, because weaker individuals will probably give up earlier (Bee 2002). In our first experiment, the alpha males were larger than the betas, and the number of advertisement calls was positively correlated with male condition. This partially corroborates our first hypothesis, and suggests that males of B. goiana may be assessing the condition of their opponents through their calls, and thus avoiding costly conflicts with large males. However, a recent study on the acoustic behavior of B. goiana showed that males did not alter their vocal activity in response to playbacks simulating a larger opponent (Morais et al. 2016), suggesting that males may not be able to assess their opponents’ size solely through their calls.

If B. goiana males do not assess the condition of their opponents via their calls to avoid unnecessary fights, what could explain the low prevalence of physical contact during contests? We suggest two non-exclusive explanations. The first relates to fighting motivation: if males had already mated before the experiments, this should decrease their motivation to engage in costly contest behaviors (Brown et al. 2007). In anurans, energetic depletion, and the resulting decrease in motivation, may occur before any measurable decrease in body condition (e.g. bullfrogs, Hettyey et al. 2009). Because we performed most of our experiments in the middle-late reproductive season (January-May), and given the constraints of in-situ experimental designs, if some males had previously mated, they are less likely to engage in costly confrontations with other males. The second explanation deals with the low frequency of scars in males: very few males used in the experiments had scars, contrasting to species such as Boana curupi (Garcia, Faivovich & Haddad, 2007), in which 90% of the males exhibited fighting scars (Candaten et al. 2020). This could mean that either B. goiana is inherently less aggressive than other gladiator frogs or, alternatively, that resources were not limited enough to motivate aggression (Arnott and Elwood 2008). For example, males of B. curupi fight to monopolize a few oviposition sites along a stream, while oviposition in B. goiana occurred along the entire stream area (Dias et al. 2017, Candaten et al. 2020). However, to further test these hypotheses, comparisons among different populations and experiments controlling for previous matings are necessary. Nonetheless, it is important to highlight that the argument that B. goiana males do not evaluate their opponent’s size via their calls comes from an experiment using playback (Morais et al. 2016). Thus, another possibility is that males of this species employ a multimodal communication, assessing their opponents using more than one type of signal, such as visual ones (Starnberger et al. 2014). Combination of acoustic and visual signals have been reported for several anuran species, including hylids closely related to B. goiana, such as Boana albomarginata (Spix, 1824) and Boana albopunctata (Spix, 1824) (Giasson and Haddad 2006, Toledo et al. 2007). These visual signals occur even in nocturnal species (Hartmann et al. 2004) and may be very subtle, such as opening the mouth or inflating the vocal sac without calling (Lipinski et al. 2012), behaviors that were observed in B. goiana during our experiments (T.M. Dias, pers. obs.). Thus, a possibility remains that physical combats were rare during our study because males were able to use more than one source of information to assess their opponents’ size to avoid unnecessary conflicts, though this hypothesis needs further testing and does not exclude other possible explanations mentioned above.

The aggressive calls of B. goiana probably have the role of maintaining a spatial separation between calling males, and thus may serve as a warning to intruders (Wells 1977a, Dyson and Passmore 1992). The reduction of the acoustic interference, achieved through the maintenance of spacing between males, may be more important for male reproductive success than the possession of a particular calling site (Whitney and Krebs 1975, Wells 1977a, Nali and Prado 2014). Resident males are also more likely to win territorial disputes, because the cost of losing a territory is higher for a resident (Wells 1978, Bee et al. 1999, Owen and Gordon 2005). Additionally, large males usually win contests against small males (Howard 1978, Wells 1978). In our second experiment, large males emitted more aggressive calls and the more males in the chorus, the larger was the distance adopted by the nearest male. This suggests that males of B. goiana can detect the spatial position of other males and that intruder males can adjust their behavior according to the social context (Wells 1977b). Thus, maintaining a suitable distance from the resident male or avoiding areas where they are calling may be strategies adopted by intruder males of B. goiana to avoid undesired physical combats and/or acoustic interference. Similar behaviors were described for another species of gladiator frog, Bokermannohyla ibitiguara (Cardoso, 1983) (Nali and Prado 2012, 2014). Interestingly, the increase in male density did not lead to an increase in aggression levels or any other change in calling behaviors (Table 1), dismissing our second hypothesis. General theory predicts that competition should become more intense the more valuable (or scarce) a resource is (Grant 1993, Arnott and Elwood 2008). Since it does not seem to be the case for B. goiana, two explanations are possible. The first is a scenario where there are too many competitors, meaning that the costs of defense are high and any potential benefit does not pay off (Grant 1993). This does not seem to be the case, because even though aggression levels were low, there was a clear establishment and defense of territories, with only two males adopting alternative strategies such as satellite behavior. The second explanation could be that resource value does not increase enough to motivate an increase in aggressive behavior. This makes sense when we consider that, in our case, the resource is a mating opportunity. Although we predicted such opportunities are restricted across both time (the reproductive season) and space (the reproductive site), the results showed that this may not be the case – there is evidence that females of B. goiana mate more than once in the same reproductive season (discussed in more details below, and see Results). If mating opportunities are not as restricted as previously thought, investing in increased aggression may not be an adaptive strategy because such behavior is costly and risky (Briffa and Sneddon 2007), and maintaining the spacing between calling males in a chorus seems to be a more cost-effective strategy, as indicated by our results discussed above (Tables 1 and 2, Figs 2, 3).

Although most males were territorial, some males behaved as satellite males (sensu Wells 2007) during the territorial experiments and this strategy was already observed for many anuran species (e.g. Toledo and Haddad 2005, Leary et al. 2005, Nali and Prado 2012). This alternative strategy has been suggested to represent an attempt of males to intercept females approaching calling males or obtain a vacant territory (Wells 1977b, Haddad 1991). Another strategy would be engaging in a fight in order to obtain the resident’s territory. However, physical combats were rare during both experiments and never exceeded 5 s (T.M. Dias, pers. obs.). Aggressive behaviors can include both aggressive calls and physical combats (Wogel et al. 2002, Costa et al. 2010 Lemes et al. 2012). However, as costs of physical combats can be high, males of B. goiana may be investing more in aggressive calls and, sometimes, adopting a satellite posture, which may be important to decrease energy expenditure and avoid injuries that could be caused by fights (Kluge 1981, Nali and Prado 2012). However, because satellite behavior only occurred in 2 of the 30 males used in the second experiment, the establishment of territories and maintenance of spacing between males mostly via calling behavior seems to be the norm for this population of B. goiana.

There was a positive relationship between the body size of males and females of B. goiana in amplexus, which indicates size-assortative mating and corroborates our third hypothesis. In some anuran species, females are able to assess male traits and choose those that might increase their fitness (Robertson 1990, Reynolds and Fitzpatrick 2007, Valencia-Aguilar et al. 2020). Size-assortative mating has been reported for some anurans and is suggested to favor a close juxtaposition of cloacae (Licht 1976, Morris 1989, Bastos and Haddad 1996, but see Green 2019). As most anurans exhibit external fertilization, better cloacae juxtaposition can maximize egg fertilization, potentially increasing the reproductive success of individuals in amplexus (Licht 1976, Robertson 1990, Bourne 1993, Bastos and Haddad 1996). In this context, it is likely that B. goiana females are choosing males with similar body sizes, though how females are evaluating the males (e.g. using acoustic signals, visual signals, or both) remains unanswered (see previous discussion regarding the males, and also Starnberger et al. 2014).

Most studies on anurans report a positive relationship between female size and clutch size/volume (e.g. Wogel et al. 2002, Dziminski and Alford 2005, Pupin et al. 2010, Guimarães et al. 2011). However, we did not find this relationship for B. goiana, and this same result was already observed for other hylid species (Lips 2001, Hartmann et al. 2004). We found that at least one female mated and spawned two times in the same reproductive season. In another occasion, after a female had spawned, we observed through the transparent skin of its abdomen that the female still carried several oocytes. These are evidences that B. goiana females may spawn more than once during each reproductive season, as observed for other anuran species (Rafínska 1991, Bastos and Haddad 1996, Vaira 2001). For species with multiple spawns, it has been suggested that the correlation between female size and size of clutches and eggs might be weak because clutches will differ in size between the beginning and the end of the breeding season (Parker and Begon 1986, Kusano and Hayashi 2002, Prado and Haddad 2005). Considering that egg size may represent a similar energetic cost for the female as the number of eggs per clutch, the same explanation could be applied for the absence of relationship between clutch size versus female size and between egg size versus eggs per clutch in this multiple-clutch species (Prado and Haddad 2005). Alternatively, the placement of the amplectant pairs in plastic bags to spawn may have affected their behavior. However, we think this is unlikely, because once ovulation begins the female is unable to prevent spawning. Thus, the observation of remaining oocytes in the abdomen of females and the lack of correlation between female size and clutch size/volume indicate that females of B. goiana spawn more than once during the reproductive season.

Our results differ in some aspects from what was previously known for B. goiana. For the population of B. goiana studied in Minas Gerais state, the mean number of eggs per clutch was higher and the mean egg size was smaller compared to our results (Menin et al. 2004). The average female SVL from the Minas Gerais population was larger than what we report here, which could explain the larger clutches found in that population (Menin et al. 2004). Furthermore, our sample size is larger and thus less likely to be affected by extreme values within the population that could affect the average values. Additionally, depending on the environmental conditions of the reproductive areas (e.g. competition, predation, food resources, unpredictability of water bodies), females may differ in the reproductive investment (e,g. fewer and larger eggs, many smaller eggs, eggs variable in size) (Crump 1984, Dziminski et al. 2009, Pupin et al. 2018). Considering the evidence for multiple spawning in B. goiana, this plastic response to dynamic environmental conditions seems plausible. Unfortunately, the available information does not allow us to infer the specific reasons for these reported differences.

We found that males were larger at the beginning of the reproductive season at our study site. Differences in the timing of male arrival may affect mating success, especially when the majority of the females arrive at the beginning of the reproductive season (Lodé et al. 2005, Wells 2007). Following this, large males arrive earlier than small males, although the reason for this is unclear (Salvador and Carrascal 1990, Lodé et al. 2005). One explanation could be that large males are older and more experienced, and thus know the reproductive area better and are able to arrive first (Reading 2001). However, this may not apply for B. goiana because no individual captured in the first season was recaptured in the second, suggesting that they may not live for more than one year. An alternative explanation could be that large males of B. goiana may simply have better physical conditions, arriving earlier at the area, and establishing territories first, as observed for Rana dalmatina Fitzinger, 1839 (Ranidae) (Lodé et al. 2005). A third explanation would be that the smaller size/condition of the males at the end of the reproductive season could reflect recruitment, as observed for Boana albopunctata (Spix, 1824) (de Sá et al. 2014). The reproductive season of B. goiana starts in October, and one possibility is that males born at the beginning of the season may already be sexually mature around May. If this is true, the entrance of these young, small males into the breeding active population may lower the average size of the males, thus explaining our results. Lastly, because resident males tend to lose weight throughout the reproductive season due to calling activity and territorial defense (Wells 1978, Robertson 1986), and may be dependent on sperm stored before the reproductive season (Hettyey et al. 2009), they may leave the chorus before the end of the breeding season. This could allow small males to enter the chorus and obtain a chance of reproducing in the late breeding season. An interesting technique that could help to discern between these explanations is to mark the tadpoles and keep track of them until maturity (e.g. Martin 2011, Courtois et al. 2013).

Males and females of B. goiana appear to be capable of adopting different reproductive strategies according to environmental conditions and social contexts. Females of this species much likely spawn multiple clutches, which can vary regarding the number of eggs and egg size. It would be particularly interesting to follow females throughout an entire reproductive season, so that we could investigate trade-offs between egg size, egg number and female investment in different environmental conditions. Females might be obtaining information about calling males, and thus selecting those with similar body sizes, as evidenced by the size-assortative mating, which could increase their fitness via increased fertilization rates. Males of B. goiana seem to avoid fights in which individuals have low probabilities to win, because physical combats were rare. This is corroborated by the distance between calling males, which increased with the number of aggressive calls of the resident male. Alternatively, low aggression levels can be the result of low motivation or even an inherent trait of B. goiana, but these hypotheses require further investigation. The possibility that this species employs multimodal communication could also explain some inconsistencies with published data, although this also requires further evaluation. Acoustic communication seems to be extremely important for B. goiana in many social contexts and future studies should investigate which information, if any, are being assessed by individuals, as well as how this information could be conveyed.

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, #476800/2011-0). We are grateful to ICMBio (Instituto Chico Mendes de Conservação da Biodiversidade) for providing the collection permits. R.P. Bastos (#304363/2010-3) and C.P.A. Prado are grateful to CNPq (#301125/2013-9), and T.M. Dias to CAPES, for fellowships. C.P.A. Prado acknowledges the Fundação de Amparo à Pesquisa do Estado de São Paulo for financial support (FAPESP #2009/12013-4). We thank P. de Marco Jr and A.V. Palaoro for the help with the statistical analyses. We are also grateful to P. Nobre, A.R. Morais, M.N. Siqueira, I. Carvalho, P.G. Gambale, and J.M. Ribeiro for help in the fieldwork; and to M.M. Dalosto for reading and commenting the manuscript, and for the help in the statistical analyses. We specially thank V.T.T. de Sousa for help in the fieldwork, statistical analyses, and for valuable insights and comments on the manuscript. The authors are also grateful to two anonymous reviewers and the editor for valuable suggestions and criticisms that greatly improved the manuscript. We dedicate this study to Marcelo Menin (in memoriam), who was a great herpetologist and a pioneer in the study of the biology of Boana goiana.